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Insights into the N-sulfation mechanism: molecular dynamics simulations of the N-sulfotransferase domain of NDST1 and mutants.

Gesteira TF, Pol-Fachin L, Coulson-Thomas VJ, Lima MA, Verli H, Nader HB - PLoS ONE (2013)

Bottom Line: Moreover, whether Lys833, His716 and Lys614 play a role in both glycan recognition and glycan sulfation remains elusive.In this study we evaluate the contribution of NDST mutants (Lys833, His716 and Lys614) to dynamical effects during sulfate transfer using comprehensive combined docking and essential dynamics.Furthermore, NDST1 mutants unveiled Lys833 as vital for both the glycan binding and subsequent N-sulfotransferase activity of NDST1.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Bioquímica, Universidade Federal de São Paulo, São Paulo, Brazil. tarsis.ferreira@cchmc.org

ABSTRACT
Sulfation patterns along glycosaminoglycan (GAG) chains dictate their functional role. The N-deacetylase N-sulfotransferase family (NDST) catalyzes the initial downstream modification of heparan sulfate and heparin chains by removing acetyl groups from subsets of N-acetylglucosamine units and, subsequently, sulfating the residual free amino groups. These enzymes transfer the sulfuryl group from 3'-phosphoadenosine-5'-phosphosulfate (PAPS), yielding sulfated sugar chains and 3'-phosphoadenosine-5'-phosphate (PAP). For the N-sulfotransferase domain of NDST1, Lys833 has been implicated to play a role in holding the substrate glycan moiety close to the PAPS cofactor. Additionally, Lys833 together with His716 interact with the sulfonate group, stabilizing the transition state. Such a role seems to be shared by Lys614 through donation of a proton to the bridging oxygen of the cofactor, thereby acting as a catalytic acid. However, the relevance of these boundary residues at the hydrophobic cleft is still unclear. Moreover, whether Lys833, His716 and Lys614 play a role in both glycan recognition and glycan sulfation remains elusive. In this study we evaluate the contribution of NDST mutants (Lys833, His716 and Lys614) to dynamical effects during sulfate transfer using comprehensive combined docking and essential dynamics. In addition, the binding location of the glycan moiety, PAPS and PAP within the active site of NDST1 throughout the sulfate transfer were determined by intermediate state analysis. Furthermore, NDST1 mutants unveiled Lys833 as vital for both the glycan binding and subsequent N-sulfotransferase activity of NDST1.

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All-atom root-mean-square deviation (RMSD) of the protein, plotted against the 50 ns MD simulation time, for the systems containing (A) the NST alone and for the (B) NST/PAPS, (C) NST/PAPS/α-GlcN-(1→4)-GlcA and (D) NST/PAP/α-GlcNS-(1→4)-GlcA complexes.Black, NST-1; Green, Lys614Ala; Blue, His716Ala, Red, Lys833Ala.
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pone-0070880-g003: All-atom root-mean-square deviation (RMSD) of the protein, plotted against the 50 ns MD simulation time, for the systems containing (A) the NST alone and for the (B) NST/PAPS, (C) NST/PAPS/α-GlcN-(1→4)-GlcA and (D) NST/PAP/α-GlcNS-(1→4)-GlcA complexes.Black, NST-1; Green, Lys614Ala; Blue, His716Ala, Red, Lys833Ala.

Mentions: Molecular Dynamics Simulation – To search for associations between local/global conformational changes and the substrate binding to the enzyme, MD simulations were performed for the complexes that resulted from docking analysis, as well as mutated, bonded and unbounded proteins. Accordingly, in order to examine conformational variations of the NST during simulations, the root-mean-square deviation (RMSD) of the Cα atomic positions with respect to the crystal structure were evaluated for the native protein and three mutants (Fig. 3). As a general feature, the obtained RMSD values achieved a plateau after the first 10 nanoseconds, with little conformational changes during their passage through plateaus. The analyses of the RMSD values of NST all-atom for the NST/PAPS complex, NST/disaccharide/PAPS complex and native enzyme alone showed that the NST/PAPS complex is relatively more stable (Fig. 3A and B), with lower RMSD fluctuations, compared to native enzyme, PAPS/α-GlcN-(1→4)-GlcA and PAP/α-GlcNS-(1→4)-GlcA complexes (Fig. 3C and D). The complex NST/PAP/α-GlcNS-(1→4)-GlcA (black) MD simulations presents a decrease in RMSD fluctuations over time due to the eventual stabilization of the substrate/enzyme complex which shifts to a stable orientation/conformation after an initial rearrangement. In order to acquire specific data on disaccharide positioning and fluctuations during the simulation, the RMSD for the disaccharide in relation to NST complexes were obtained based on the MD simulations. The RMSD of α-GlcN-(1→4)-GlcA atoms rose to 2.0 Å after 3 ns, presenting fluctuating peaks with this maximum amplitude during the entire simulation, indicating that an equilibrium state is not achieved for the non-sulfated moiety during the simulation in the presence of PAPS (Fig. S3). This fluctuation on RMSD is also observed using an octasaccharide as ligand (data not shown). Interestingly, the RMSD values for the mutant models, although increased, were more stable, reflecting the influence of these residues in the enzyme catalysis (Fig. 3C and D). Time-dependent secondary structure fluctuations were analyzed using the DSSP program [20], and most of the secondary structures (such as the β-sheet and α-helix) from the initial structure remained stable (Fig. S4a–d).


Insights into the N-sulfation mechanism: molecular dynamics simulations of the N-sulfotransferase domain of NDST1 and mutants.

Gesteira TF, Pol-Fachin L, Coulson-Thomas VJ, Lima MA, Verli H, Nader HB - PLoS ONE (2013)

All-atom root-mean-square deviation (RMSD) of the protein, plotted against the 50 ns MD simulation time, for the systems containing (A) the NST alone and for the (B) NST/PAPS, (C) NST/PAPS/α-GlcN-(1→4)-GlcA and (D) NST/PAP/α-GlcNS-(1→4)-GlcA complexes.Black, NST-1; Green, Lys614Ala; Blue, His716Ala, Red, Lys833Ala.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3733922&req=5

pone-0070880-g003: All-atom root-mean-square deviation (RMSD) of the protein, plotted against the 50 ns MD simulation time, for the systems containing (A) the NST alone and for the (B) NST/PAPS, (C) NST/PAPS/α-GlcN-(1→4)-GlcA and (D) NST/PAP/α-GlcNS-(1→4)-GlcA complexes.Black, NST-1; Green, Lys614Ala; Blue, His716Ala, Red, Lys833Ala.
Mentions: Molecular Dynamics Simulation – To search for associations between local/global conformational changes and the substrate binding to the enzyme, MD simulations were performed for the complexes that resulted from docking analysis, as well as mutated, bonded and unbounded proteins. Accordingly, in order to examine conformational variations of the NST during simulations, the root-mean-square deviation (RMSD) of the Cα atomic positions with respect to the crystal structure were evaluated for the native protein and three mutants (Fig. 3). As a general feature, the obtained RMSD values achieved a plateau after the first 10 nanoseconds, with little conformational changes during their passage through plateaus. The analyses of the RMSD values of NST all-atom for the NST/PAPS complex, NST/disaccharide/PAPS complex and native enzyme alone showed that the NST/PAPS complex is relatively more stable (Fig. 3A and B), with lower RMSD fluctuations, compared to native enzyme, PAPS/α-GlcN-(1→4)-GlcA and PAP/α-GlcNS-(1→4)-GlcA complexes (Fig. 3C and D). The complex NST/PAP/α-GlcNS-(1→4)-GlcA (black) MD simulations presents a decrease in RMSD fluctuations over time due to the eventual stabilization of the substrate/enzyme complex which shifts to a stable orientation/conformation after an initial rearrangement. In order to acquire specific data on disaccharide positioning and fluctuations during the simulation, the RMSD for the disaccharide in relation to NST complexes were obtained based on the MD simulations. The RMSD of α-GlcN-(1→4)-GlcA atoms rose to 2.0 Å after 3 ns, presenting fluctuating peaks with this maximum amplitude during the entire simulation, indicating that an equilibrium state is not achieved for the non-sulfated moiety during the simulation in the presence of PAPS (Fig. S3). This fluctuation on RMSD is also observed using an octasaccharide as ligand (data not shown). Interestingly, the RMSD values for the mutant models, although increased, were more stable, reflecting the influence of these residues in the enzyme catalysis (Fig. 3C and D). Time-dependent secondary structure fluctuations were analyzed using the DSSP program [20], and most of the secondary structures (such as the β-sheet and α-helix) from the initial structure remained stable (Fig. S4a–d).

Bottom Line: Moreover, whether Lys833, His716 and Lys614 play a role in both glycan recognition and glycan sulfation remains elusive.In this study we evaluate the contribution of NDST mutants (Lys833, His716 and Lys614) to dynamical effects during sulfate transfer using comprehensive combined docking and essential dynamics.Furthermore, NDST1 mutants unveiled Lys833 as vital for both the glycan binding and subsequent N-sulfotransferase activity of NDST1.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Bioquímica, Universidade Federal de São Paulo, São Paulo, Brazil. tarsis.ferreira@cchmc.org

ABSTRACT
Sulfation patterns along glycosaminoglycan (GAG) chains dictate their functional role. The N-deacetylase N-sulfotransferase family (NDST) catalyzes the initial downstream modification of heparan sulfate and heparin chains by removing acetyl groups from subsets of N-acetylglucosamine units and, subsequently, sulfating the residual free amino groups. These enzymes transfer the sulfuryl group from 3'-phosphoadenosine-5'-phosphosulfate (PAPS), yielding sulfated sugar chains and 3'-phosphoadenosine-5'-phosphate (PAP). For the N-sulfotransferase domain of NDST1, Lys833 has been implicated to play a role in holding the substrate glycan moiety close to the PAPS cofactor. Additionally, Lys833 together with His716 interact with the sulfonate group, stabilizing the transition state. Such a role seems to be shared by Lys614 through donation of a proton to the bridging oxygen of the cofactor, thereby acting as a catalytic acid. However, the relevance of these boundary residues at the hydrophobic cleft is still unclear. Moreover, whether Lys833, His716 and Lys614 play a role in both glycan recognition and glycan sulfation remains elusive. In this study we evaluate the contribution of NDST mutants (Lys833, His716 and Lys614) to dynamical effects during sulfate transfer using comprehensive combined docking and essential dynamics. In addition, the binding location of the glycan moiety, PAPS and PAP within the active site of NDST1 throughout the sulfate transfer were determined by intermediate state analysis. Furthermore, NDST1 mutants unveiled Lys833 as vital for both the glycan binding and subsequent N-sulfotransferase activity of NDST1.

Show MeSH
Related in: MedlinePlus